Ang II plays a pivotal role in chronic cardiac disease, however, its signaling pathways leading to cardiac fibrosis remain largely unclear. It is known that Ang II acts by stimulating TGF-b to mediate cardiac fibrosis, however, our preliminary studies found that Ang II is able to directly activate the TGF-b signaling pathway by two mechanisms: 1) an acute pathway (5-30 minutes) via activation of the ERK/p38 MAP kinases. This is TGF-b-independent since Ang II is able to activate Smad2 &3 in cells lacking TGF-b receptors; this response is blocked by ERK or p38 inhibitors; 2) a late mechanism (24 hours) that acts through autocrine TGF-b and leads to fibrosis. Furthermore, we also found that mice null for SmadS are protected against cardiac fibrosis, while mice that are conditionally deleted for Smad2 enhance fibrosis in response to Ang II. Thus, we hypothesize that Smad signaling is a key to the development of cardiac fibrosis in response to Ang II. We plan to test this hypothesis by pursuing three specific aims. In Specific Aim 1, we propose to identify new signaling pathways whereby Ang II mediates cardiac fibrosis. We will demonstrate that Ang II signals through the AT1-R and activates an acute Smad signaling via the ERK/p38 MAPK-dependent mechanism (5-30 mins). We also propose to identify that a long-term effect of Ang II by activating a late Smad signaling via the classic TGF-b-dependent mechanism (24hrs). In Specific Aim 2, we will dissect the specific role of Smad2 or SmadS in Ang ll-mediated fibrosis in mouse embryonic fibroblasts lacking Smad2 or SmadS and in cardiac fibroblasts that do not express SmadS or have conditional knockout for Smad2. In Specific Aim 3, we will further investigate the functional role of Smad2 or SmadS in cardiac fibrosis in mice null for SmadS KO mice or have conditional KO for Smad2 by subcutaneous infusion of large doses of Ang II. We expect that the outcomes obtained will support the central hypothesis, providing new insights into the pathogenesis of Ang ll-mediated cardiac fibrosis and information for the development of new therapeutic strategies. [unreadable] [unreadable]